JP2009507771A - Cancerous disease modifying antibodies - Google Patents

Abstract

The present invention relates to methods for producing cancerous disease modifying antibodies in patients using the novel paradigm of screening. By isolating anti-cancer antibodies using the cytotoxicity of cancer cells as an endpoint, this method allows the production of anti-cancer antibodies for therapeutic and diagnostic purposes. These antibodies can be used as an aid in cancer staging and diagnosis and can be used to treat primary tumors and tumor metastases. Anti-cancer antibodies can be conjugated to toxins, enzymes, radioactive compounds and hematogenous cells.
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Description

(Refer to related applications)
This application claims the benefit of the filing date of provisional application 60 / 704,646 filed August 2, 2005, the contents of which are incorporated herein by reference.

  The present invention relates to the isolation and production of cancerous disease modifying antibodies (CDMAB) and their use in therapeutic and diagnostic processes, optionally in combination with one or more chemotherapeutic agents. The present invention further relates to binding assays utilizing the CDMAB of the present invention.

  Monoclonal antibodies as cancer treatments: Each individual who exhibits cancer is unique and has a cancer that is different from other cancers as well as the individual's uniqueness. Nevertheless, current treatment treats all patients with the same type and stage of cancer in the same way. At least 30 percent of these patients fail primary therapy, thus leading to a further series of treatments, increasing the chances of treatment failure, metastasis, and eventually death. An excellent method of treatment is a special specification of treatment in a particular individual. The only current treatment that in itself leads to special specifications is surgery. Chemotherapy and radiation therapy cannot be tailored to the patient, and the surgery itself is in most cases insufficient to cause healing.

  With the advent of monoclonal antibodies, the possibility of developing custom-made treatment methods becomes more realistic, since each antibody can be directed to a single epitope. Moreover, it is possible to generate antibody combinations that are directed to a panel of epitopes that uniquely define a particular individual's tumor.

  With the recognition that the significant difference between cancerous and normal cells is that the cancerous cells contain antigens that are specific for the transfected cells, the scientific community specifically binds to the cancer antigens Have long thought that monoclonal antibodies can be designed to specifically target transfected cells, and therefore monoclonal antibodies serve as a “magic bullet” to eliminate cancer cells Created a belief that However, it is now widely recognized that there is no single monoclonal antibody that can serve in all cases of cancer, and that monoclonal antibodies can be deployed as a class of targeted cancer therapy. Monoclonal antibodies isolated according to the disclosed teachings of the present invention have been shown to modify the process of cancerous disease, for example, in a way that benefits the patient by reducing tumor burden. Various references are made herein as modified antibodies (CDMAB) or “anti-cancer” antibodies.

  At present, cancer patients generally have few treatment options. Strictly controlled approaches to cancer treatment have led to improvements in global survival and morbidity. However, for certain individuals, these improved statistics do not necessarily correlate with improvements in their personal condition.

  Thus, when a methodology is proposed that allows a practitioner to treat each tumor independently of other patients in the same cohort, it allows for a unique treatment approach that is adapted for just one person. Become. Ideally, if such a treatment process would increase the rate of healing and produce better outcomes, it would meet the long-standing need.

  Historically, the use of polyclonal antibodies has been used with limited success in human cancer therapy. Lymphoma and leukemia have been treated with human plasma, but there has been little long-term remission or response. Furthermore, there was a lack of reproducibility and no additional benefit compared to chemotherapy. Solid tumors such as breast cancer, melanoma and renal cell carcinoma were also treated with human blood, chimpanzee serum, human plasma and horse serum with similarly unpredictable and ineffective results.

  Many clinical trials of monoclonal antibodies have been conducted in solid tumors. In the 1980s, at least four clinical trials were conducted in human breast cancer, using antibodies to specific antigens or based on tissue selectivity, and only 1 out of 47 patients responded . In 1998, a clinical trial using a humanized anti-Her2 / neu antibody (Herceptin®) in combination with cisplatin was successful. In this study, responses were evaluated in 37 patients, approximately one-quarter had a partial response rate, and one-fourth had a slight or stable disease progression. The median response time for responders was 8.4 months, and the median response duration was 5.3 months.

  Herceptin® was approved in 1998 for primary use in combination with Taxol®. The results of the clinical study showed that the disease progressed in the group (6.9 months) ingested antibody treatment + Taxol (registered trademark) compared to the group ingested Taxol (registered trademark) alone (3.0 months) It was shown that the median time spent increased. There was also a slight increase in median survival, with the Herceptin + Taxol® treatment group versus the Taxol® alone treatment group 22 months versus 18 months. In addition, numbers increased in both the complete (8 percent vs. 2 percent) and partial responders (34 percent vs. 15 percent) in the antibody + Taxol ® combination group compared to Taxol ® alone. However, treatment with Herceptin® and Taxol® resulted in a higher incidence of cardiotoxicity compared to treatment with Taxol® alone (13 percent vs. 1 percent, respectively). Herceptin® therapy also overexpresses human epidermal growth factor receptor 2 (Her2 / neu), a receptor that does not have a currently known function or biologically important ligand (immune / immune). It was only effective for patients (determined by histochemical (IHC) analysis) and was approximately 25 percent of patients with metastatic breast cancer. Thus, there is still a great unmet need in breast cancer patients. Even patients who can benefit from Herceptin® treatment still require chemotherapy and therefore still have to address at least some of the side effects of this type of treatment.

  Clinical trials investigating colorectal cancer involve antibodies against both glycoprotein and glycolipid targets. For antibodies such as 17-1A with some specificity for adenocarcinoma, phase 2 clinical trials were conducted in 60 patients and only one patient had a partial response. In another study, the use of 17-1A was only one complete response and only two minor responses out of 52 patients in a protocol using additional cyclophosphamide. To date, a phase III clinical trial of 17-1A has not demonstrated improved efficacy as an adjunct therapy for stage III colon cancer. The use of a humanized murine monoclonal antibody that was initially approved for imaging also did not result in tumor regression.

  More recently, positive results have been obtained in colorectal cancer clinical studies using monoclonal antibodies. In 2004, ERBITUX® was approved for secondary treatment in patients with EGFR-expressing metastatic colorectal cancer that is refractory to irinotecan-based chemotherapy. The results of both the Group 2 phase II clinical trial and the single group study showed that ERBITUX® combined with irinotecan had a median disease progression time of 4.1 months and 6.5 months respectively. It was shown to have a reaction rate of 23 percent and 15 percent. The results of the same group 2 phase II clinical trial and another single group study showed that treatment with ERBITUX® alone had a median disease progression time of 1.5 months and 4.2 months, respectively. It was shown to result in 11 percent and 9 percent reaction rates, respectively.

  Thus, treatment of ERBITUX® in combination with irinotecan in both Switzerland and the United States, and treatment of ERBITUX® alone in the United States, is a second treatment for patients with colon cancer who have failed primary irinotecan therapy. Approved as the next treatment. Therefore, like Herceptin®, treatment in Switzerland is only approved as a combination of monoclonal antibodies and chemotherapy. In addition, treatment in both Switzerland and the United States is only approved as a second-line therapy for patients. Also in 2004, AVASTIN® was approved for use in combination with intravenous 5-fluorouracil-based chemotherapy as the primary treatment for metastatic colorectal cancer. The results of a phase III clinical study demonstrated that the median survival of patients treated with AVASTIN® + 5-fluorouracil was prolonged compared to patients treated with 5-fluorouracil alone (20 each, Months vs. 16 months). However, again, like Herceptin® and ERBITUX®, treatment is only approved as a combination of monoclonal antibody and chemotherapy.

  It also continues to produce poor results in lung, brain, ovary, pancreas, prostate and stomach cancer. The most recent promising result of non-small cell lung cancer is that the monoclonal antibody (SGN-15; dox-BR96, anti-Sialyl-LeX) treatment binds to the cell killing agent doxorubicin in combination with the chemotherapeutic agent TAXOTERE®. ) From a Phase II clinical trial. TAXOTERE® is the only FDA-approved chemotherapy for secondary treatment of lung cancer. Initial data shows overall improved survival compared to TAXOTERE® alone. Of the 62 patients mobilized for the study, two-thirds took SGN-15 in combination with TAXOTERE®, while the remaining one third was TAXOTERE®. Ingested alone. In patients taking SGN-15 in combination with TAXOTERE®, the median overall survival was 7.3 compared to 5.9 months for patients taking TAXOTERE® alone. Months. The overall survival at 1 year and 18 months for each patient who took SGN-15 + TAXOTERE® was 24% and 8% respectively for those who took TAXOTERE® alone. 29 percent and 18 percent. Further clinical trials are planned.

  In preclinical use of monoclonal antibodies in melanoma has had some limited success. Few of these antibodies have reached clinical trials, and to date, none have been approved or demonstrated favorable results in Phase III clinical trials.

  The discovery of new drugs to treat the disease has been hampered by the lack of identifying relevant targets among the 30,000 products of known genes that clearly contribute to the pathogenesis of the disease. In oncology studies, potential drug targets are often selected by the fact that they are simply overexpressed in tumor cells. The targets thus identified are then screened for interaction with a number of compounds. In potential antibody therapy, these candidate compounds are typically traditional in the production of monoclonal antibodies according to the basic principles established by Kohler and Milstein (1975, Nature, 256, 495-497, Kohler and Milstein). Induced by various methods. Spleen cells are collected from mice immunized with an antigen (eg, whole cells, cell fraction, purified antigen) and fused with an immortalized hybridoma partner. The resulting hybridomas are screened and selected for secretion of antibodies that bind most enthusiastically to the target. Many therapeutic and diagnostic antibodies directed against cancer cells, including Herceptin® and RITUXIMAB, have been produced using these methods and have been selected based on their affinity. There are two parts to this strategy. First, the selection of appropriate targets for therapeutic or diagnostic antibody binding is due to the lack of knowledge surrounding the tissue-specific carcinogenic course and the resulting simple identification of these targets. Limited by simple methods such as selection by overexpression. Second, the assumption that a drug molecule that binds to a receptor with the greatest affinity usually has the highest probability of initiating or inhibiting the signal may not always be the case.

  Despite some progress in the treatment of breast and colon cancer, the identification and development of effective antibody therapies is insufficient for all types of cancer, either alone or in combination.

Conventional patents:
US Pat. No. 6,057,056 discloses a method of transfecting cells from a patient's tumor with an MHC gene that can be cloned from the patient's cells or tissue. These transfected cells are then used to vaccinate the patient.

  Patent Document 2 describes a step of obtaining a monoclonal antibody that is specific for the inner cell components of mammalian new cells and normal cells but not the outer component, a step of labeling the monoclonal antibody, a labeled antibody, and killing the new cells A method comprising: contacting a tissue of a treated animal to be treated; and determining the effect of the treatment by measuring the binding of the labeled antibody to an internal cellular component of the degenerated neoplastic cell. To prepare antibodies directed against human intracellular antigens, the patent owner recognizes that malignant cells represent a convenient source of such antigens.

  Patent Document 3 provides a novel antibody and a method for producing the same. In particular, the patent teaches the formation of monoclonal antibodies that bind strongly to protein antigens associated with human tumors, such as colon and lung tumors, but have the property of binding to a much lesser extent to normal cells.

  Patent Document 4 describes surgical removal of tumor tissue from human cancer patients, treatment of tumor tissue to obtain tumor cells, irradiation of tumor cells that are alive but non-tumorigenic, The present invention provides a method for treating cancer comprising preparing a vaccine capable of suppressing recurrence of a primary tumor and simultaneously suppressing translocation for the patient. This patent teaches the development of monoclonal antibodies reactive with tumor cell surface antigens. As described in column 4, line 45 (see below), the patent owner utilizes spontaneous tumor cells to develop monoclonal antibodies that express specific immunotherapy active in human neoplasia.

  U.S. Patent No. 6,057,031 teaches a glycoprotein antigen that has the characteristics of a human carcinoma but does not depend on the epithelial cells of origin.

  Patent Document 6 describes an anti-Her2 antibody that induces apoptosis of Her2-expressing cells, a hybridoma cell line that produces the antibody, a method of cancer treatment using the antibody, and a pharmaceutical composition comprising the antibody.

  U.S. Patent No. 6,057,031 describes a novel hybridoma cell line for producing monoclonal antibodies against mucin antigens purified from tumor and non-tumor tissue sources.

  U.S. Patent No. 6,057,031 describes a method of generating human lymphocytes that produce antibodies specific to the desired antigen, a method of producing monoclonal antibodies, and a monoclonal antibody produced by this method. This patent specifically describes the production of anti-HD human monoclonal antibodies useful for the diagnosis and treatment of cancer.

  Patent document 9 relates to an antibody, an antibody fragment, an antibody complex, and a single-chain immunotoxin that are reactive to human carcinoma cells. There are two parts to the mechanism by which these antibodies function: the molecule reacts with cell membrane antigens present on the surface of human carcinomas, and the antibodies are incorporated into the carcinoma cells and subsequently bound. And is particularly useful for forming antibody-drug and antibody-toxin conjugates. In unmodified form, the antibody also exhibits cytotoxic properties at specific concentrations.

U.S. Patent No. 6,057,031 discloses the use of autoantibodies for tumor treatment and prevention. However, this antibody is an antinuclear autoantibody from an aged mammal. In this case, autoantibodies are said to be one type of natural antibody found in the immune system. Since the autoantibody is from an “old mammal”, there is no requirement that the autoantibody is from a patient that is actually being treated. In addition, this patent discloses natural and monoclonal antinuclear autoantibodies from aged mammals and hybridoma cell lines that produce monoclonal antinuclear autoantibodies.
US Pat. No. 5,750,102 U.S. Pat. No. 4,861,581 US Pat. No. 5,171,665 US Pat. No. 5,484,596 US Pat. No. 5,693,763 US Pat. No. 5,783,186 US Pat. No. 5,849,876 US Pat. No. 5,869,268 US Pat. No. 5,869,045 US Pat. No. 5,780,033

  This application utilizes the methodology for producing patient-specific anti-cancer antibodies taught in US Pat. No. 6,180,357 for isolating hybridoma cell lines encoding cancerous disease modifying monoclonal antibodies. To do. These antibodies can be made specifically for a single tumor, and thus can make cancer treatments special. Within the context of this application, an anti-cancer antibody having either cell killing properties (cytotoxicity) or cell growth inhibiting properties (cytostatic properties) is referred to hereinafter as cytotoxicity. These antibodies can be used as an aid in cancer staging and diagnosis and can be used to treat tumor metastases. These antibodies can also be used for cancer prevention in a prophylactic treatment manner. Unlike antibodies generated according to traditional drug discovery paradigms, antibodies generated by this method target molecules and pathways that have not previously been shown as integral to malignant tissue growth and / or survival. can do. Furthermore, the binding affinity of these antibodies is suitable for the requirements for the initiation of cytotoxic events that may not follow stronger affinity interactions. It is also within the scope of the present invention to combine standard chemotherapeutic modalities, such as radionuclides, and CDMAB of the present invention, thereby focusing on the use of said chemotherapeutic agent. CDMAB can also bind to toxins, cytotoxic moieties, enzymes such as biotin-conjugated enzymes or hematogenous cells, thereby forming antibody conjugates.

  Personalized anti-cancer therapeutic prospects will change the way patients are managed. A likely clinical scenario is to obtain and store a tumor sample at the time of diagnosis. From this sample, tumors can be classified by a panel of cancerous disease modifying antibodies already present. Patients are staged as before, but available antibodies can be used to further classify the patient. Patients can be readily treated with existing antibodies, and a panel of tumor specific antibodies can be generated using the methods outlined herein or in conjunction with the screening methods disclosed herein. It can be produced either by using a display library. The generated antibodies are all added to the library of anti-cancer antibodies, since other tumors may have some of the same epitopes being treated. Antibodies produced according to the methods of the invention can be useful for treating cancerous diseases in any number of patients with cancers that bind to these antibodies.

  In addition to anti-cancer antibodies, patients can choose to receive current recommended treatment as part of a variety of treatment regimens. Due to the fact that antibodies isolated by the methodology of the present invention are relatively non-toxic to non-cancerous cells, it is possible to use high dose antibody combinations alone or in conjunction with conventional therapies. enable. A high therapeutic index also allows for a short period of retreatment that should reduce the likelihood of the development of treatment resistant cells.

  If the patient is refractory to the initial course of treatment or metastasis occurs, the method of generating antibodies specific for the tumor can be repeated for re-treatment. In addition, anti-cancer antibodies can be combined with red blood cells obtained from the patient and reinjected for treatment of metastases. There are few effective treatments for metastatic cancer, and metastasis is usually a precursor to a poor outcome leading to death. However, metastatic cancers are usually well vascularized and delivery of anti-cancer antibodies by erythrocytes can have the effect of concentrating the antibodies to the tumor site. Even before metastasis, most cancer cells depend on the host's blood supply for survival, and anti-cancer antibodies bound to red blood cells can be effective against in situ tumors. Alternatively, the antibody can be conjugated to other hematopoietic cells such as lymphocytes, macrophages, monocytes, natural killer cells and the like.

  There are five types of antibodies, each associated with a function conferred by its heavy chain. In general, cancer cell killing by naked antibodies is thought to be mediated by either antibody-dependent cytotoxic activity or complement-dependent cytotoxicity. For example, murine IgM and IgG2a antibodies can activate human complement by binding the C-1 component of the complement system, thereby leading to the classical pathway of complement activation that can result in oncolysis. Can be activated. For human antibodies, the most effective complement active antibodies are generally IgM and IgG1. IgG2a and IgG3 isotype murine antibodies are effective in mobilizing cytotoxic cells with Fc receptors that lead to cell death by monocytes, macrophages, granulocytes and certain lymphocytes. Both IgG1 and IgG3 isotypes of the human antibody mediate ADCC.

  Another possible mechanism of antibody-mediated cancer death is through the use of so-called catalytic antibodies, which function to catalyze the hydrolysis of various chemical bonds in cell membranes and their associated glycoproteins or glycolipids. sell.

  There are three additional mechanisms for antibody-mediated cancer cell killing. The first is the use of antibodies as vaccines that induce the body to generate an immune response against putative antigens present in cancer cells. The second is the use of antibodies that target the growth receptor and interfere with its function or down-regulate its receptor so that function is effectively lost. Third, such antibodies to direct ligation of cell surface moieties that can result in direct cell death, such as ligation of death receptors such as TRAIL R1 or TRAIL R2, or integrin molecules such as alpha Vbeta3. It is an effect.

The clinical usefulness of anticancer drugs is based on the benefit of the drug under the patient's acceptable risk profile. In cancer therapy, survival is generally the most pursued benefit, but there are many other well-recognized benefits in addition to prolonging life. These other benefits where treatment does not have a deleterious effect on survival include symptom relief, protection against adverse events, increased time to relapse or disease-free survival, and increased time to progression. These criteria are generally accepted, and regulatory agencies such as the US Food and Drug Administration (FDA) have approved drugs that produce these benefits (Hirschfeld et al. Critical Reviews in Oncology / Hematology 42: 137). -143 2002). In addition to these criteria, it is well recognized that there are other endpoints that can foresee these types of benefits. In part, the accelerated approval method approved by the US FDA acknowledges that there are surrogates that are likely to predict patient benefit. At the end of the year (2003), 16 drugs were approved by this method and 4 of them were fully approved, ie, follow-up would provide direct patient benefits predicted by the surrogate endpoint. Demonstrated. One important endpoint in determining drug effects in solid tumors is to assess tumor burden by measuring response to treatment (Therase et al. Journal of the National Cancer Institute 92 (3): 205-216. 2000). Clinical criteria for such evaluation (RECIST criteria) have been published by the Response Evaluation Criteria in Solid Tumors Working Group, an international group of cancer specialists. Agents that have demonstrated an effect on tumor burden, as demonstrated by objective responses according to the RECIST criteria, compared to appropriate control groups, tend to ultimately produce direct patient benefit. In preclinical settings, tumor burden is generally more direct to assessment and documentation. Agents that produce increased survival in preclinical models have the greatest predictive clinical utility because preclinical studies can be converted to clinical settings. Similar to producing a positive response to clinical treatment, drugs that reduce tumor burden in the preclinical setting can also have a significant direct impact on the disease. Although extended survival is the clinical outcome most sought after in anticancer drug treatment, there are other benefits that have clinical utility, such as tumor burden that can be correlated with delayed disease progression, extended survival, or both. reduction, lead to direct benefits, it is evident that it is also possible to provide a clinical effect (Eckhardt et al.Developmental Therapeutics: Successes and Failures of clinical Trial Designs of Targeted Compounds; ASCO Educational Book, 39 th Annual Meeting , 2003, pages 209-219).

  The present invention describes the development and use of AR59A816.14 whose effects are identified by cytotoxicity assays and in animal models of human cancer. The present invention is a reagent that specifically binds to an epitope or a plurality of epitopes present in a target molecule, and has a property of in vitro cytotoxicity against malignant tumor cells as a naked antibody, No reagent, and as a naked antibody, a reagent that directly mediates inhibition of tumor growth is described. A further advance is the use of such anti-cancer antibodies targeting tumors expressing cognate antigen markers to achieve tumor growth inhibition and other positive endpoints for cancer treatment.

  In all of these, the present invention teaches the use of the AR59A816.14 antigen as a therapeutic target that can reduce the tumor burden of a cancer that expresses the antigen in a mammal when administered. The present invention also targets CDMAB (AR59A816.14), derivatives thereof, antigen-binding fragments thereof, and cytotoxicity thereof, to reduce the tumor burden of cancers that express the antigen in mammals. Teaches the use of activity-inducing ligands. The present invention further teaches the use of detecting the AR59A816.14 antigen in cancerous cells, which is useful for the diagnosis, treatment prediction and prognosis of mammals with tumors that express this antigen. be able to.

  Accordingly, in order to isolate hybridoma cell lines and corresponding isolated monoclonal antibodies and antigen-binding fragments thereof encoded by said hybridoma cell lines, cancerous cells from one particular individual or one or more specific cancers Produces cancerous disease modifying antibodies (CDMAB) raised against cell lines, where CDMAB is cytotoxic with respect to cancer cells but at the same time is relatively nontoxic to non-cancerous cells It is an object of the present invention to utilize the method.

  It is an additional object of the invention to teach cancerous disease modifying antibodies, and ligands and antigen binding fragments thereof.

  It is a further object of the present invention to produce cancerous disease modifying antibodies whose cytotoxicity is mediated by antibody-dependent cytotoxicity.

  It is still an additional objective of the present invention to produce cancerous disease modifying antibodies whose cytotoxicity is mediated by complement dependent cytotoxicity.

  It is a still further object of the present invention to produce cancerous disease modifying antibodies whose cytotoxicity is a function of their ability to catalyze the hydrolysis of cellular chemical bonds.

  A still further object of the present invention is to produce cancerous disease modifying antibodies that are useful in binding assays for cancer diagnosis, prognosis and monitoring.

  Other objects and advantages of the present invention will become apparent from the following description, and specific embodiments of the present invention will be described by way of illustration and example.

(Brief description of the drawings)
The patent or application file contains at least one drawing made in color. Copies of this patent or patent application publication in color drawings will be provided by the JPO upon request and payment of the necessary fees.
FIG. 1 compares the cytotoxicity and binding level rates of hybridoma supernatants against cell lines MDA-MB-231, OVCAR-3, SW1116, Lovo and CCD-27sk.
FIG. 2 represents the binding of AR59A816.14 and anti-EGFR control to cancer and normal cell lines. Data are summarized in a table representing the mean fluorescence intensity as an increase in folding over the isotype control.
FIG. 3 includes representative FACS histograms of AR59A816.14 and anti-EGFR antibodies directed against several cancer and non-cancer cell lines.
FIG. 4 shows the effect of AR59A816.14 on tumor growth in a prophylactic Lovo colon cancer model. The vertical line indicates the period during which the antibody was administered. Data points represent the mean +/- SEM.
FIG. 5 shows the effect of AR59A816.14 on body weight in a prophylactic Lovo colon cancer model. Data points represent the mean +/- SEM.
FIG. 6 shows the effect of AR59A816.14 on tumor growth in a prophylactic DLD-1 colon cancer model. The vertical line indicates the period during which the antibody was administered. Data points represent the mean +/- SEM.
FIG. 7 shows the effect of AR59A816.14 on body weight in a prophylactic DLD-1 colon cancer model. Data points represent the mean +/- SEM.
FIG. 8 tabulates an IHC comparison of AR59A816.14 compared to positive and negative controls against human xenograft tumor tissue.
9A, 9B, 9C and 9D show AR59A816.14 or xenograft MDA-MB-231 (C) or colon SW1116 (D) for xenograft tumor tissue of breast MDA-MB-231 (A) or colon SW1116 (B). ) Is a representative photomicrograph showing the binding pattern of the buffer control to the xenograft tumor tissue. AR59A816.14 showed positive staining in tumor cells. The magnification is 400 ×.
FIG. 10 tabulates the IHC comparison of AR59A816.14 compared to positive and negative controls for human liver tumor microarrays.
11A, 11B, 11C, and 11D show AR59A816.14 for primary (A) versus metastatic (B) HCC tumor tissue, or buffer control for primary (C) versus metastatic (D) HCC tumor tissue. It is a typical micrograph which shows the coupling | bonding pattern. AR59A816.14 showed positive staining for metastasis at a higher rate compared to primary HCC samples. The magnification is 200 ×.

  In general, the following words or phrases have the indicated definitions when used in the disclosure, description, examples and claims of the invention.

  The term “antibody” is used in the broadest sense and specifically includes, for example, single monoclonal antibodies (including agonists, antagonists and neutralizing antibodies, deimmunized, murine, chimerized or humanized antibodies), poly Covering antibody compositions, single chain antibodies, immune complexes and antibody fragments with epitope specificities (see below).

  As used herein, the term “monoclonal antibody” means an antibody obtained from a population of substantially homogeneous antibodies, ie, individual antibodies contained in a population may be present in small amounts. Identical except for the possibility of naturally occurring mutations. Monoclonal antibodies are highly specific and are directed to a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to these specificities, monoclonal antibodies are advantageous in that they can be synthesized without being contaminated with other antibodies. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous antibody population, and should not be construed as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies used according to the present invention can be obtained from Kohler et al. , Nature, 256: 495 (1975), or can be made by the hybridoma (murine or human) method or see recombinant DNA methods (eg, see US Pat. No. 4,816,567). ). “Monoclonal antibodies” are described, for example, in Clackson et al. , Nature, 352: 624-628 (1991) and Marks et al. , J .; MoI. Biol, 222: 581-597 (1991) can also be used to isolate from a phage antibody library.

“Antibody fragments” comprise a portion of an intact antibody, preferably comprising the antigen binding or variable region. Examples of antibody fragments include less than full length antibodies, Fab, Fab ′, F (ab ′) ₂ and Fv fragments; bispecific antibodies; linear antibodies; single chain antibody molecules; single chain antibodies, single domains Examples include multispecific antibodies formed from antibody molecules, fusion proteins, recombinant proteins, and antibody fragments.

An “intact” antibody is one that includes an antigen binding variable region and further includes a light chain constant region domain (C _L ) and a heavy chain constant region domain, C _H 1, C _H 2 and C _H 3. The constant region domain can be a native sequence constant region domain (eg, a human native sequence constant region domain) or an amino acid sequence variable region. Preferably, the intact antibody has one or more effector functions.

  Depending on the amino acid sequence of the constant region domain of the heavy chain, intact antibodies can be assigned to different “classes”. These are the five main classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, some of which are further “subclassed” (isotypes), eg, IgG1, IgG2, IgG3, It can be divided into IgG4, IgA and IgA2. The heavy chain constant region domains that correspond to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

  The “effector function” of an antibody means a biological activity that contributes to the Fc region (native sequence Fc region or amino acid sequence variable region Fc region) of the antibody. Examples of antibody effector functions include Clq binding; complement dependent cytotoxicity; Fc receptor binding; antibody dependent cell mediated cytotoxicity (ADCC); phagocytosis; cell surface receptors (eg, B cell receptors; BCR ) Downward control.

  “Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to non-specific cytotoxic cells that express Fc receptors (FcR) (eg, natural killer (NK) cells, neutrophils and macrophages) It refers to a cell-mediated reaction that recognizes the above bound antibody and subsequently causes lysis of the target cell. NK cells, the primary cells that mediate ADCC, express FcγRIII only, whereas monocytes express FcγRI, FcγRII and FcγRIII. A summary of FcR expression in hematopoietic cells is described in Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991), page 464, Table 3. To assess the ADCC activity of the molecule of interest, an in vitro ADCC assay as described in US Pat. Nos. 5,500,362 or 5,821,337 can be performed. Effector cells useful for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest is determined in vivo, eg, Clynes et al. It can be evaluated in an animal model such as that disclosed in PNAS (USA) 95: 652-656 (1998).

  “Effector cells” are leukocytes that express one or more FcRs and perform effector functions. Preferably, the cell expresses at least FcγRIII and performs ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils, with PBMC and NK cells being preferred. Effector cells can be isolated from their native source, eg, from blood or PBMC as described herein.

  The terms “Fc receptor” or “FcR” are used to describe a receptor that binds to the Fe region of an antibody. A preferred FcR is a native sequence human FcR. Furthermore, preferred FcRs are those that bind to IgG antibodies (gamma receptors), including FcγRI, FcγRII and FcγRIII subclasses of receptors including allelic variants of these receptors and alternative splice forms. FcγRIII receptors include FcγRIIA (“activating receptor”) and FcγRIIB (“inhibiting receptor”), which have similar amino acid sequences that differ primarily in the cytoplasmic domain. Activating receptor FcγRIIA contains an immunoreceptor tyrosine activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine inhibition motif (ITIM) in its cytoplasmic domain (see review of M. in Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). FcR is described in Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991); Capel et al. , Immunomethods 4: 25-34 (1994); and de Haas et ah, J. Am. Lab. Clin. Med. 126: 330-41 (1995). Other FcRs, including those identified in the future, are encompassed by the term “FcR” herein. This term also includes the neonatal receptor FcRn involved in the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., Eur. J. Immunol. 24). : 2429 (1994)).

  “Complement dependent cytotoxicity” or “CDC” means the ability of a molecule to lyse a target in the presence of complement.The complement activation pathway involves the first component of the complement system (Clq), Initiated by binding to a molecule (eg, an antibody) that is complexed with a cognate antigen To assess complement activation, see, for example, Gazzano-Santoro et al., J. Immunol. 163 (1996) can be performed.

  The term “variable region” refers to the fact that certain portions of a variable region of an antibody vary greatly in sequence and are used for the binding and specificity of each specific antibody to a specific antigen. . However, variability is not evenly distributed throughout the variable region domains of antibodies. It is concentrated in three segments called hypervariable region in both light and heavy chain variable region domains. The more highly conserved portions of variable region domains are called the framework region (FR). The variable region domains of native heavy and light chains are each joined by three hypervariable region regions, forming a loop that is connected to the> sheet structure, and in some cases forms part of it. It includes four FRs that are generally β-sheet shaped. The hypervariable region in each chain is held together closer to the FR and together with the hypervariable region of the other chain contributes to the formation of the antigen binding site of the antibody (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. Pp 15-17; 48-53 (1991)). The constant domain is not directly involved in the binding of the antibody to the antigen, but exhibits various effector functions such as antibody participation in antibody-dependent cellular cytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the amino acid residues of an antibody which are responsible for antigen binding. The hypervariable region is generally an amino acid residue from a “complementarity determining region” or “CDR” (eg, residues 24-34 (L1), 50-56 (L2) and 89-97 in the light chain variable region domain). (L3), residues 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health. Service, National Institutes of Health, Bethesda, Md. Pp 15-17; 48-53 (1991)) and / or residues from the “hypervariable region loop” (residue 2632 (L1) in the light chain variable region domain, 50-52 (L2) and 91-96 (L3), residues 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Less J. MoI. Biol. 196: 901 917 (1987)). “Framework Region” or “FR] residues are those variable domain domain residues other than the hypervariable region residues as defined herein.Papain digestion of antibodies is referred to as the“ Fab ”fragment 2 Produces two identical antigen-binding fragments, each with a single antigen-binding site and a residue “Fc” fragment whose name reflects the ability to easily crystallize. Pepsin treatment yields an F (ab ′) ₂ fragment that has two antigen binding sites and is still capable of cross-linking antigen.

“Fv” is the minimum antibody fragment that contains a complete antigen recognition and antigen binding site. This region is composed of a dimer of one heavy chain and one light chain variable region domain in close non-covalent association. It is this arrangement that the three hypervariable region of each variable domain interact to define the antigen binding site on the surface of the V _H -V _L dimer. Collectively, the six hypervariable region confers antigen binding specificity to the antibody. However, even a single variable region domain (or half Fv containing only three hypervariable region regions specific for the antigen) has the ability to recognize and bind antigen, but with more affinity than the entire binding site. The nature is low. The Fab fragment also contains the constant region domain of the light chain and the first constant region domain (CH1) of the heavy chain. Fab ′ fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab′-SH is the name herein for Fab ′ in which the cysteine residues of the constant domain have at least one free thiol group. F (ab ′) ₂ antibody fragments were originally produced as a pair of Fab ′ fragments with a hinge cysteine between them. Other chimeric linkages of antibody fragments are also known.

  The “light chain” of an antibody from any vertebrate species is one of two distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain. Can be assigned to.

“Single-chain Fv” or “scFv” antibody fragments comprise the V _H and V _L domains of antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the _VH and _VL domains that is capable of forming the scFv into the desired structure for antigen binding. A review of scFv can be found in Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).

The term “bispecific antibody” is a small antibody fragment having two antigen binding sites, the fragment being bound to the variable region light domain (V _L ) in the same polypeptide chain (V _H -V _L ). Contains the variable region heavy domain (V _H ). By using a linker that is too short to allow pairing between two domains on the same chain, the domain can pair with the complementary domain of another chain to create two antigen-binding sites. Forced. Bispecific antibodies are described, for example, in EP 404,097; WO 93/11161; and Hollinger et al, Proc. Natl. Acad. Sci USA, 90: 6444-6448 (1993).

  An “isolated” antibody is one that has been identified, separated and / or recovered from a component of its natural environment. Natural environmental contaminants are substances that interfere with the diagnostic or therapeutic use of antibodies, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

  An antibody that “binds” to an antigen of interest is one that can bind to the antigen with sufficient affinity such that the antibody is useful as a therapeutic or diagnostic agent that targets cells that express the antigen. is there. If the antibody binds to an antigenic part, it usually binds preferentially to the antigenic part rather than to other receptors and is common for accidental binding such as non-specific Fc contacts or for other antigens. It does not include those that bind to post-translational modifications that are targeted, and may not significantly cross-react with other proteins. Methods for detecting antibodies that bind to the antigen of interest are well known in the art and can include, but are not limited to, FACS, cell ELISA and Western blot.

  As used herein, the expressions “cell”, “cell line” and “cell culture” are used interchangeably, and all such names include progeny. It is also understood that all progeny may not be exactly identical in DNA content due to intentional or accidental mutations. Mutant progeny that have the same function or biological activity as screened on the original transformed cells are included. It is clear from the context that a clear name is intended.

  “Treatment” refers to both therapeutic treatment and prophylactic or preventative measures, the purpose being to prevent or delay (reduce) the targeted pathological condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to having the disorder or those in which the disorder must be prevented. Accordingly, the mammal being treated herein may be diagnosed as having a disorder, or may be susceptible or susceptible to a disorder.

  The terms “cancer” and “cancerous” refer to or describe the pathological condition in mammals that is typically characterized by unregulated cell growth or death. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma and leukemia or lymphoid malignancy. More particularly, examples of such cancers include squamous cell carcinoma (eg, epithelial squamous cell carcinoma), small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung cancer including squamous cell carcinoma of the lung, peritoneum Cancer, hepatocellular carcinoma, stomach or abdominal cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal Cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, anal cancer, penile cancer, and head and neck cancer.

  A “chemotherapeutic agent” is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN ™); alkyl sulfonates such as busulfan, improsulfan and piperosulfan; such as benzodopa, carbocon, metredorpa and ureadopa Aziridines; altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphaoramide, and ethyleneimine and methylellamelamine including trimethylololomelamine; chlorambucil, chlornafazine, colophosphamide, estramustine, ifosfamide , Meclomethamine, oxidized mecloethamine hydrochloride, melphalan, nobenbitine, phenesterine, prednisomes Nitrogen mustards such as guanosine, trophosphamide, uracil mustard; Nitrosourea such as camulustin, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; acracinomycin, actinomycin, auramycin, azaserine, bleomycin, cactinomycin, caliomycin Thiamycin, carabicin, carnomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenol Acid, nogaramycin, olivomycin, peplomycin, podophylomycin, puromycin, keramycin, rhomycin Antibiotics such as rubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); like denopterin, methotrexate, pteropterin, trimethrexate Folic acid analogues; purine analogues such as fludarabine, 6-mercaptopurine, thiampurine, thioguanine; such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxyfluridine, enocitabine, floxuridine, 5-FU Androgen such as carsterone, drmostanolone propionate, epithiostanol, mepithiostan, test lactone; aminoglutethi Anti-adrenal drugs such as mid, mitotan, trilostane; folic acid supplements such as florin; acegraton; aldophosphaamide glycosides; aminolevulinic acid; amsacrine; vestlabcil; bisantrene; edatraxate; defofamine; demecorsin; Eleptinium acetate; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenmet; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; Schizophyllan; spirogermanium; tenuazonic acid; triadicon; 2,2 ', 2 "-trichlorotriethylamine; urethane; vindesine; Carbazine; mannomustine; mitoblonitol; mitactol; piperobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes such as paclitaxel (TAXOL <(R)>, Bristol-Myers Squibb Oncology, Nc. J. et al. ) And docetaxel (TAXOTERE®, Aventis, Rhone-Poulenc Roller, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; (VP-16); Ifosfamide; Mitomycin C; Mitoxantrone; Vincristine; Vinorelbine; Navelbine; Novantrone; Teniposide; Daunomycin; Aminopterin; Retinoic acid; esperamicin; capecitabine; and any of the above drugs Acceptable salt, acid, or derivatives. Also included in this definition are, for example, antiestrogens including tamoxifen, raloxifene, aromatase inhibition 4 (5) -imidazole, 4-hydroxy tamoxifen, trioxyphene, keoxifene, LY11018, onapristone and toremifene (Fareston). Antihormonal agents that act to modulate or inhibit hormonal effects on tumors; antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and any of the pharmaceutically acceptable salts, acids, Or a derivative.

  “Mammals” for therapeutic purposes are mammals including humans, mice, SCID or nude mice or mouse strains, livestock, zoo animals, sporting animals or pets such as sheep, dogs, horses, cats, cows, etc. Means any animal classified as an animal. Preferably, the mammal herein is a human.

  “Oligonucleotides” can be prepared using the solid phase technique described in EP 266,032 published May 4, 1988, or by Froehler et al, Nucl. Acids Res. 14: 5399-5407, 1986, the length chemically synthesized by known methods (eg phosphotriester, phosphite or phosphoramidite chemistry) via the deoxynucleoside H-phosphonate intermediate described in Short single or double stranded polydeoxynucleotides. These are then purified on a polyacrylamide gel.

  A “chimeric” antibody is an antibody whose heavy and / or light chain portions are derived from a particular species or belong to a particular antibody class or subclass as long as they exhibit the desired biological activity. An antibody that is identical or homologous to the corresponding sequence, while the remaining chains are derived from another species or belong to another antibody class or subclass, and of such antibodies It is an immunoglobulin that is identical or homologous to the corresponding sequence in the fragment (US Pat. No. 4,816,567 and Morrison et al, Proc. Natl. Acad. ScL USA, 81: 6851-6855 (1984). )).

“Humanized” forms of non-human (eg, murine) antibodies are specific chimeric immunoglobulins, immunoglobulin chains or fragments thereof (eg, Fv, Fab, Fab ′) that contain minimal sequence derived from non-human immunoglobulin. , F (ab) ₂ or other antigen binding subsequence of an antibody) In most cases, humanized antibodies are non-human species such as mice, rats or rabbits in which the complementarity determining region (CDR) residues of the recipient antibody have the desired specificity, affinity and ability ( It is a human immunoglobulin (recipient antibody) that has been replaced with the CDR residues of the donor antibody. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced with corresponding non-human FR residues. Furthermore, humanized antibodies can comprise residues that are found neither in the recipient antibody nor in the foreign CDR or FR sequences. These modifications are further purified to optimize antibody performance. In general, humanized antibodies comprise substantially all of at least one, typically two variable region domains, all or substantially all CDR regions corresponding to non-human immunoglobulin, and all or substantially. All FR residues are of human immunoglobulin consensus sequence. A humanized antibody optimally also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

  A “deimmunized” antibody is an immunoglobulin that is non-immunogenic or less immunogenic to a given species. Deimmunization can be achieved by structural modifications to the antibody. Any deimmunization technique known to those skilled in the art can be used. One suitable technique for deimmunizing antibodies is described, for example, in WO 00/34317 published 15 June 2000.

Antibodies that induce “apoptosis” include, but are not limited to, Annexin V binding, caspase activity, DNA fragmentation, cell shrinkage, endoplasmic reticulum expansion, cell fragmentation and / or membrane vesicles (with apoptotic bodies). Induced programmed cell death by any means exemplified by the formation of

  Throughout this specification, hybridoma cell lines, as well as isolated monoclonal antibodies produced therefrom, are alternatively referred to as the internal name AR59A816.14 or the deposit name IDAC170505-02.

As used herein, “antibody ligand” includes a moiety that exhibits specific binding to at least one epitope of a target antigen, an intact antibody molecule, an antibody fragment, and at least an antigen binding region or portion thereof. Any molecule having (ie, the variable region portion of an antibody molecule), eg, Fv molecule, Fab molecule, Fab ′ molecule, F (ab ′) ₂ molecule, bispecific antibody, fusion protein, or IDAC 170505-02 Any genetically engineered molecule that specifically recognizes and binds to at least one epitope of an antigen (IDAC 170505-02 antigen) that is bound to an isolated monoclonal antibody produced by a hybridoma cell line .

  As used herein, “cancerous disease modifying antibodies” (CDMAB) are cancerous by methods that benefit patients, for example, by reducing tumor burden or extending the survival of individuals with tumors. It refers to monoclonal antibodies and their antibody ligands that modify the process of sexually transmitted diseases.

  As used herein, “antigen binding region” means the part of a molecule that recognizes a target antigen.

  As used herein, “competitively inhibit” refers to a monoclonal antibody (IDAC 170505-02 antibody) produced by a hybridoma cell line called IDAC 170505-02, using a conventional mutual antibody competition assay. It means that the determinant being directed can be recognized and bound to it. (Belanger L., Sylvestre C. and Dufour D. (1973), Enzyme linked immunoassay for alpha fetiotein by compandive and sanda procedure. C. 48).

  As used herein, a “target antigen” is the IDAC 170505-02 antigen or portion thereof.

  As used herein, an “immunoconjugate” is any antibody, such as an antibody that is chemically or biologically conjugated to a cytotoxin, radioactive agent, enzyme, toxin, antitumor agent or therapeutic agent. Or CDMAB. The antibody or CDMAB can be bound to any position along the molecule as long as it can bind to its target to the cytotoxin, radioactive agent, antitumor agent or therapeutic agent. Examples of immune complexes include antibody toxin chemical complexes and antibody toxin fusion proteins.

  As used herein, “fusion protein” means any chimeric protein in which an antigen binding region is bound to a biologically active molecule, such as a toxin, enzyme or protein drug.

  In order that the invention described herein may be more fully understood, the following description is set forth.

  The present invention provides CDMAB that specifically recognizes and binds to the IDAC 170505-02 antigen (ie, IDAC 170505-02 CDMAB).

  CDMAB, an isolated monoclonal antibody produced by a hybridoma deposited at IDAC with accession number 170505-02, competes for immunospecific binding of the isolated monoclonal antibody produced by hybridoma IDAC 170505-02 to its target antigen. As long as it has an antigen-binding region that inhibits, any form can be used. Accordingly, any recombinant protein having the same binding specificity as the IDAC 170505-02 antibody (eg, a fusion protein in which the antibody is combined with a second protein such as lymphokine or tumor inhibitory growth factor) is within the scope of the invention. .

  In one embodiment of the invention, the CDMAB is an IDAC 170505-02 antibody.

In another embodiment, the CDMAB is an antigen binding fragment, which comprises an Fv molecule (eg, a single chain Fv molecule), a Fab molecule, a Fab ′ molecule, a F (ab ′) ₂ molecule, a fusion protein, a bispecific antibody. , A heterologous antibody, or any recombinant molecule having the antigen binding region of the IDAC 170505-02 antibody. The CDMAB of the invention is directed to the epitope to which the IDAC 170505-02 monoclonal antibody is directed.

  The CDMAB of the present invention can be modified to produce derivative molecules, i.e., modified by intramolecular amino acid modifications. Chemical modification may also be possible.

  Derivative molecules retain the functional properties of the polypeptide, ie, molecules with such substitutions still allow the polypeptide to bind to the IDAC 170505-02 antigen or portion thereof.

  These amino acid substitutions include, but are not limited to, amino acid substitutions known in the art as “conservative”.

  For example, certain amino acid substitutions, referred to as “conservative amino acid substitutions,” are well-established protein chemistry that can be made frequently without altering either protein conformation or function in the protein. The principle.

  Such changes include isoleucine (I), valine (V) and leucine (L) with one of the other hydrophobic amino acids, aspartic acid (D) with glutamic acid (E) (and vice versa). ), Glutamine (Q) with asparagine (N) (and vice versa), and serine (S) with threonine (T) (and vice versa). Other substitutions can also be considered conservative depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) can be frequently exchanged, as are alanine and valine (V). Methionine (M), which is relatively hydrophobic, can be frequently exchanged with leucine and isoleucine and frequently for valine. Lysine (K) and arginine (R) can frequently swap positions, which is that the significant feature of amino acid residues is their charge, and it is not significant that the pK of these two amino acid residues is different Because there is no. Still other changes can be considered “conservative” under certain circumstances.

  Upon obtaining the antibody, one of ordinary skill in the art can generate a competitively inhibiting CDMAB, eg, a competing antibody, which recognizes the same epitope (Belanger L et al. Clinica Chimica Acta 48:15). -18 (1973)). One method involves immunizing with an immunogen that expresses the antigen recognized by the antibody. Samples can include, but are not limited to, tissues, isolated proteins or cell lines. The resulting hybridomas can be screened using a competition assay that identifies antibodies that inhibit test antibody binding, such as ELISA, FACS, or Western blot. Another method can be performed by using a phage display antibody library and panning antibodies that recognize at least one epitope of the antigen (Rubinstein JL et al. Anal Biochem 314: 294-300 (2003)). ). In either case, the antibody is selected based on its ability to overcome the binding of the initially labeled antibody to at least one epitope of its target antigen. Thus, such antibodies have the characteristic of recognizing at least one epitope of the antigen, like the original antibody.

Example 1
Hybridoma production-hybridoma cell line AR59A816.14
Hybridoma cell line AR59A816.14 was obtained in accordance with the Budapest Treaty. Deposited on May 17th. In accordance with 37 CFR 1.808, the depositor confirms that all restrictions imposed on the public availability of the deposited material are irrevocably removed upon grant of the patent. The deposit will be replaced if the depository is unable to give a live sample.

  To produce a hybridoma producing the anti-cancer antibody AR59A816.14, a single cell suspension of frozen human colon metastatic liver tissue (Genomics Collaborative, Cambridge, Mass.) Was prepared in PBS. IMMUNEASY ™ (Qiagen, Venlo, Netherlands) adjuvant was prepared for use by gentle mixing. 5-7 week old BALB / c mice were immunized by subcutaneous injection of 2 million cells in 50 microliters of antigen adjuvant. Immunized mice were boosted intraperitoneally with 2 million cells in 50 microliters using freshly prepared antigen adjuvant 2 and 5 weeks after the initial immunization. Three days after the last immunization, the spleen was used for fusion. Hybridomas were prepared by fusing isolated splenocytes with NSO-1 myeloma partners. Supernatants of the fusion were tested on hybridoma subclones.

An ELISA assay was used to determine if the antibody secreted by the hybridoma cells is an IgG or IgM isotype. Concentration of 2.4 micrograms / mL goat anti-mouse IgG + IgM in 100 microliters / well 4 ° C. coating buffer (0.1 M carbonate / bicarbonate buffer, pH 9.2-9.6) H + L) was added to the ELISA plate overnight. Plates were washed 3 times with wash buffer (PBS + 0.05 percent Tween). 100 microliters / well blocking buffer (5 percent milk in wash buffer) was added to the plate and incubated for 1 hour at room temperature, then washed 3 times with wash buffer. 100 microliters / well of hybridoma supernatant was added and the plates were incubated for 1 hour at room temperature. Plates were washed 3 times with wash buffer and diluted 1 / 100,000 in either 100 microliters / well of goat anti-mouse IgG or IgM horseradish peroxidase complex (diluted in PBS containing 5 percent milk). Added). After incubating the plate for 1 hour at room temperature, the plate was washed 3 times with wash buffer. 100 microliters / well of TMB solution was incubated for 1-3 minutes at room temperature. The color reaction, 50 was terminated by addition of 2M _H 2 SO ₄ microliters / well, the plates were a Perkin-Elmer HTS7000 plate reader and read at 450nm using a reference filter of 595 nm. As shown in FIG. 1, the AR59A816.14 hybridoma cell line secreted mainly IgG isotype antibodies.

  To determine the subclass of antibodies secreted by hybridoma cells, isotyping experiments were performed using the Mouse Monoclonal Antibody Isotyping Kit (GE Healthcare, Baie d'Urfe, QC). Antibody-containing hybridoma supernatants were added to test tubes (in a 1:10 dilution with TBS-T) having isotyped strips carrying goat antibodies specific for different types of peptide chains. The tube was stirred for 15 minutes. The strip was then washed twice with TBS-T for 5 minutes with agitation. Peroxidase-labeled species-specific anti-mouse antibody was added to a test tube (in 1: 500 dilution with TBS-T) for 15 minutes to detect monoclonal antibody bound to the goat antibody on the stick. The strip was again washed twice with TBS-T for 5 minutes with agitation. The peroxidase labeled antibody bound to the strip was then detected using a peroxidase substrate system. 30 mg of 4-chloro-1-naphthol tablets were dissolved in 10 mL of cold ethanol and a drop of hydrogen peroxide solution (30 percent v / v) was diluted with 50 mL of TBS. The two solutions were combined just prior to use and 3 mL was added to the isotyped strip with stirring for 15 minutes. The substrate solution was then discarded and the strip was washed 3 times with stirring with 5 mL distilled water. The typed stick was then removed from the test tube and the results analyzed. Anticancer antibody AR59A816.14 is of the IgG2a, κ isotype.

After a series of limiting dilutions, the hybridoma supernatants were tested for antibodies that bind to the target cells by a cell ELISA assay. One human breast cancer cell line, one ovarian cancer cell line, two human colon cancer cell lines and one human normal skin cell line were tested, MDA-MB-231, OVCAR-3, SW1116, Lovo and CCD, respectively. -27sk. All cell lines were obtained from the American Type Tissue Collection (ATCC; Manassas, Va.). Plated cells were fixed before use. Plates were washed 3 times at room temperature with PBS containing MgCl ₂ and CaCl ₂ . 100 microliters of 2 percent paraformaldehyde diluted in PBS was added to each well for 10 minutes at room temperature and then discarded. The plate was again washed 3 times with PBS containing MgCl ₂ and CaCl ₂ at room temperature. Blocking was performed with 100 microliters / well of 5% milk in wash buffer (PBS + 0.05 percent Tween) and incubated for 1 hour at room temperature. Plates were washed 3 times with wash buffer and hybridoma supernatant was added at 75 microliters / well and incubated for 1 hour at room temperature. Plates were washed 3 times with wash buffer and diluted at 1 / 25,000 dilution of goat anti-mouse IgG or IgM antibody conjugated to horseradish peroxidase at 100 microliters / well (PBS diluted with 5 percent milk). ) Was added. After 1 hour incubation at room temperature, the plates were washed 3 times with wash buffer and 100 microliters / well TMB substrate was incubated for 1-3 minutes at room temperature. The reaction was terminated by 2M _H 2 SO ₄ of 50 microliters / well, the plates were a Perkin-Elmer HTS7000 plate reader and read at 450nm using a reference filter of 595 nm. The results summarized in the table in FIG. 1 are expressed as the number of folds over background compared to the in-house IgG isotype control previously shown to not bind to the cell lines tested. The hybridoma AR59A816.14 antibody showed the highest binding to both the colon cancer cell lines SW1116 and Lovo, followed by the ovarian cancer cell line OVCAR-3. AR59A816.14 showed no detectable binding to breast cancer cell line MDA-MB-231 or normal skin cell line CCD-27sk.

Along with testing for antibody binding, the cytotoxic effect of the hybridoma supernatant was tested in cell lines MDA-MB-231, OVCAR-3, SW1116, Lovo and CCD-27sk. Calcein AM was obtained from Molecular Probes (Eugene, OR) and the assay was performed as outlined below. Cells were plated at a predetermined appropriate density prior to assay. Two days later, 75 microliters of the hybridoma microtiter plate supernatant was transferred to a cell plate and incubated for 5 days in a 5 percent CO ₂ incubator. Wells serving as positive controls are aspirated until empty and 100 microliters of sodium azide (NaN ₃ , 0.01 percent, Sigma, Oakville, ON), cycloheximide (CHX, 0.5 micromolar, Sigma, Oakville, ON) or anti-EGFR antibody (c225, IgG1, kappa, 5 microgram / mL, Cedarlane, Hornby, ON). After 5 days of treatment, the plate was inverted and emptied and blotted dry. Room temperature DPBS (Dulbecco's phosphate buffered saline) containing MgCl ₂ and CaCl ₂ was dispensed from each multichannel squeeze bottle into each well, tapped 3 times, inverted to empty, then blotted dry. Fifty microliters of fluorescent calcein dye diluted with DPBS containing MgCl ₂ and CaCl ₂ was added to each well and incubated at 37 ° C. in a 5 percent CO ₂ incubator for 30 minutes. The plate was read on a Perkin-Elmer HTS7000 fluorescent plate reader and the data was analyzed with Microsoft Excel.

The results are summarized in the table in FIG. The supernatant of the AR59A816.14 hybridoma is specifically cytotoxic in 14 percent of OVCAR-3 cells, which is 20 percent and 40 percent cytotoxicity obtained with the positive controls sodium azide and cycloheximide, respectively. Met. As summarized in the table in FIG. 1, AR59A816.14 did not cause cytotoxicity in CCD-27sk normal cells. The results in FIG. 1 demonstrated that the cytotoxic effect of AR59A816.14 was not related to the level of binding to each cancer cell type. Cytotoxicity was only observed in OVCAR-3 cells, while the highest binding was seen in colon cell lines. Known non-specific cytotoxic agents cycloheximide and NaN ₃ generally produced cytotoxicity as expected. Anti-EGFR antibody c225 produced cytotoxicity to SW1116 as expected.

Example 2
In vitro binding AR59A816.14 monoclonal antibody was produced by culturing hybridomas in CL-1000 flasks (BD Biosciences, Oakville, ON). Supernatant collection and revaccination were performed twice a week. Standard antibody purification procedures according to Protein G Sepharose 4 Fast Flow (Amersham Biosciences, Baie d'Urfe, QC) were followed. It is within the scope of the invention to utilize a deimmunized, humanized, chimerized or murine monoclonal antibody.

Binding of AR59A816.14 to breast (MDA-MB-231), colon (DLD-1, Lovo, SW620 and SW1116), prostate (PC-3) cancer cell lines, and skin (CCD-27sk) and lung Binding of (Hs888.Lu) to non-cancer cell lines was assessed by flow cytometry (FACS). All cell lines were obtained from the American Type Tissue Collection (ATCC; Manassas, Va.). Cells were prepared for FACS by first washing the cell monolayer with DPBS (without Ca ⁺⁺ and Mg ⁺⁺ ). Cells were then removed from the cell culture plate at 37 ° C. using cell dissociation buffer (INVITROGEN, Burlington, ON). After centrifugation and collection, the cells are resuspended in DPBS (staining medium) containing MgCl ₂ , CaCl ₂ and 2 percent fetal calf serum at 4 ° C., counted, aliquoted to the appropriate cell density, and centrifuged. Cells were then pelleted and resuspended in ice in 20 microgram / mL staining medium at 4 ° C. in the presence of test antibody (AR59A816.14) or control antibody (isotype control, anti-EGFR) for 30 minutes. Prior to the addition of Alexa Fluor 546-conjugated secondary antibody, the cells were washed once with staining medium. Alexa Fluor 546-conjugated antibody in staining medium was then added at 4 ° C. for 30 minutes. The cells were then finally washed and resuspended in fixing medium (staining medium containing 1.5 percent paraformaldehyde). Cell flow cytometry acquisition was assessed by sampling the FACSarray ™ using a FACSarray ™ System Software (BD Biosciences, Oakville, ON). Cell forward scatter (FSC) and side scatter (SSC) were set by adjusting the voltage and amplitude gain of the FSC and SSC detectors. The detector for the fluorescent (Alexa-546) channel was adjusted by applying unstained cells so that the cells had a uniform peak with a median fluorescence intensity of approximately 1-5 units. For each sample, approximately 10,000 gated events (stained fixed cells) were obtained for analysis and the results are presented in FIG.

  FIG. 2 tabulates the mean increase in fluorescence intensity folding over the isotype control. A representative histogram of the AR59A816.14 antibody is summarized in FIG. AR59A816.14 showed the strongest binding to colon cancer cell lines DLD-1 (201.7 fold), Lovo (143.2 fold) and SW620 (147.5 fold). Strong binding was also observed in the SW1116 colon cancer cell line (46.2 times) and the prostate cancer line PC-3 (38.5 times). Weaker binding was seen in the breast cancer cell line MDA-MB-231 (5.4 times). AR59A816.14 is a normal lung and skin cell line, Hs888. It did not bind to Lu and CCD-27sk. These data demonstrated that AR59A816.14 binds to different cancer cell lines and has the highest degree of binding to colon cancer. AR59A816.14 also demonstrated selectivity in binding since no detectable binding was detected against both human normal cell lines. These results are consistent with those obtained in Example 1.

Example 3
In vivo tumor experiments with Lovo cells Examples 1 and 2 demonstrated that AR59A816.14 has anti-cancer properties against cancer cell lines and demonstrated the highest binding to colon cell types. The antibodies were then tested in an in vivo model of human colon cancer. Referring to FIGS. 4 and 5, 4-6 week old female SCID mice were implanted with 1 million human colon cancer cells (Lovo) in 100 microliters of saline injected subcutaneously into the neck of the neck. Mice were randomly divided into 5 treatment groups in two. On the day of transplantation, 20 mg / kg of AR59A816.14 test antibody or buffer control is diluted from the stock concentrate with a diluent containing 2.7 mM KCl, 1 mM KH ₂ PO ₄ , 137 mM NaCl and 20 mM Na ₂ HPO _4. And then intraperitoneally administered to each cohort in a volume of 300 microliters. The antibody and control samples were then administered by the same method once a week for 7 weeks. Tumor growth was measured with calipers approximately every 7 days up to 8 weeks or until individual animals reached the Canadian Council for Animal Care (CCAC) endpoint. Animal weights were recorded once a week during the study. At the end of the study, all animals were euthanized according to CCAC guidelines.

  AR59A816.14 prevented tumor growth and reduced tumor burden in a preventive in vivo model of human colon cancer. On day 56 after transplantation, ie 6 days after the last treatment dose, the mean tumor volume in the AR59A816.14 treatment group was 4 percent of that in the buffer control treatment group (p <0.0016, t− Test, FIG. 4).

  In xenograft tumor models, body weight is often used as a surrogate indicator of disease progression. As can be seen in FIG. 5, there was no significant difference in body weight between groups at the end of the treatment period (p = 0.2947, t-test). Within each group, the average body weight of the animals did not change significantly at the beginning and end of the study period (buffer treatment group, p = 0.0752, t-test; AR59A816.14 treatment group, p = 1.000).

  Thus, AR59A816.14 was well tolerated and reduced tumor burden in a human colon cancer xenograft model.

Example 4
In vivo tumor experiments with DLD-1 cells The results of Example 3 were expanded to different models of human colon cancer. Referring to FIGS. 6 and 7, 4-6 week old female SCID mice were injected with 5 million human colon cancer cells (DLD-1) in 100 microliters of saline subcutaneously in the neck of the neck. Transplanted. Mice were randomly divided into 5 treatment groups in two. On the day of transplantation, 20 mg / kg of AR59A816.14 test antibody or buffer control is diluted from the stock concentrate with a diluent containing 2.7 mM KCl, 1 mM KH ₂ PO ₄ , 137 mM NaCl and 20 mM Na ₂ HPO _4. And then intraperitoneally administered to each cohort in a volume of 300 microliters. The antibody and control samples were then administered once a week by the same method for the duration of the study. Tumor growth was measured with calipers approximately every 7 days. The study was terminated after 7 injections (48 days) because the animals reached the Canadian Council for Animal Care (CCAC) endpoint due to large ulcerated lesions. Animal weights were recorded once a week during the study. At the end of the study, all animals were euthanized according to CCAC guidelines.

  AR59A816.14 prevented tumor growth and reduced tumor burden in a preventive in vivo model of human colon cancer. On the 48th day after transplantation, ie 5 days after the last treatment administration, the mean tumor volume of the AR59A816.14 treatment group was 22 percent less than that of the buffer control treatment group (FIG. 6). The tumor burden of AR59A816.14 treated mice was less than control treated mice, but not significant in this aggressive model of human colon cancer (p = 0.66, t-test). Average tumor volume at the final time point was affected by the loss of mice due to ulcerated lesions prior to the end of the treatment period. On day 27, when all mice were still alive, AR59A816.14 reduced tumor volume by 70 percent (p = 0.0002). This marked decrease was observed after only 4 injections of antibody.

  Body weight measured at weekly intervals is a substitute for health and growth failure. As seen in FIG. 7, there was no significant difference in body weight between the control treatment group and the AR59A816.14 antibody treatment group during the study period. Between groups, the average body weight of animals did not change significantly during the study period.

  AR59A816.14 was well tolerated and reduced tumor burden in a second colon cancer xenograft model.

Example 5
Staining of human tumor xenografts An IHC study was performed to characterize the distribution of AR59A816.14 antigen in human xenografts. IHC optimization studies showed that a concentration of 5 micrograms / mL with the use of proteolytic enzyme digestion was the best condition for IHC staining of AR59A816.14. Tumor xenografts were harvested from SCID mice housed in an animal facility laboratory at the Toronto General Hospital. Mice were injected with either colon cancer cell line SW1116, breast cancer cell line MDA-MB-231, MCF-7 or prostate cancer cell line LnCap, PC-3. Cell lines were obtained from the American Type Tissue Collection (ATCC; Manassas, Va.). Tumors were fixed in formalin and sent to Toronto General Hospital's Pathology Research Program Laboratory for processing. Next, a xenograft tissue microarray was constructed.

  The tissue sections were dried in an oven at 58 ° C. for 15 minutes, and dewaxed by immersing in xylene 5 times for 4 minutes each. After treatment with a series of stepwise ethanol washes (100 to 75 percent), the sections were rehydrated in water. Tissue sections were then incubated with protein-degrading enzymes (Dako, Toronto, Ontario) for 3 minutes at room temperature and washed 3 times for 5 minutes each with PBS. The sections were then immersed in 3 percent hydrogen peroxide solution for 10 minutes, washed 3 times with PBS for 5 minutes each, dried, and incubated with Universal blocking solution (Dako, Toronto, Ontario) for 5 minutes at room temperature. AR59A816.14 was diluted to a treatment concentration (5 microgram / mL) with an antibody dilution buffer (Dako, Toronto, Ontario). Cytokeratin-7 was used as a positive antibody control (ready to use; Dako, Toronto, Ontario) and antibody dilution buffer (Dako, Toronto, Ontario) was used as a negative control. The primary antibody was incubated for 1 hour at room temperature. Slides were washed 3 times with PBS for 5 minutes each. The immunoreactivity of the primary antibody was detected / visualized with a HRP-conjugated secondary antibody (ready to use; Dako Envision System, Toronto, Ontario) for 30 minutes at room temperature. After this step, the slides were washed 3 times for 5 minutes each with PBS and DAB (3,3'-diaminobenzidine tetrahydrachloride, Dako, Toronto, Ontario) chromogenic substrate solution was washed at room temperature for immunoperoxidase staining. Added for 10 minutes to cause a color reaction. The slide was washed with tap water to stop the color reaction. After counterstaining with Meyer's hematoxylin (Sigma Diagnostics, Oakville, ON), the slides were dehydrated with graded ethanol (75 to 100 percent) and clarified with xylene. The slide was covered with a coverslip using a loading medium (Dako Faramount, Toronto, Ontario). The slides were examined microscopically using Axiovert 200 (Ziess Canada, Toronto, ON), digital images were obtained and stored using Northern Eclipse Imaging Software (Mississauga, ON). The results were read, evaluated and interpreted by a histopathologist.

  The binding of the antibody to human xenografts (breast cancer MDA-MB-231 and MCF-7; prostate cancer LnCap and PC-3; and colon cancer SW1116) is moderate to MCF-MB-231. 7, strong binding to LnCap, SW1116 and PC-3 was shown (FIG. 8). Binding was limited to tumor cells. Cell localization was membrane and had a diffuse staining pattern (FIG. 9). The number of positive cells was 50 percent with MDA-MB-231 and over 50 percent with other xenografts. These results confirm what was observed in vitro by FACS in Example 2 for the MDA-MB-231, PC-3 and SW1116 cell lines, and the expression of the antigen was maintained in vivo as a xenograft. Demonstrate that

Example 6
Staining of human liver tumor tissue To assess the binding of AR59A816.14 to human liver tumor tissue, antibodies were tested on a liver tumor tissue array (lmgenex, San Diego, CA). The following information was obtained from each patient: age, gender, organ and diagnosis. The staining procedure used was the same as that disclosed in Example 5. AR59A816.14 or a negative isotype control antibody (directed to Aspergillus niger glucose oxidase (Dako, Toronto, Ontario), an enzyme that is not present or induced in mammalian tissue) is added to an antibody dilution buffer (Dako, Toronto) , Ontario) to its treatment concentration (5 microgram / mL) and cysteine-7 was used as a positive control antibody (ready to use; Dako, Toronto, Ontario).

  As disclosed in FIG. 10, AR59A816.1 showed positive binding in 28/50 (56 percent) of liver cancer sections, mainly binding to metastatic hepatocellular carcinoma, metastatic and primary Hepatocellular carcinoma was 5/8 (63 percent) vs. 19/38 (50 percent, respectively. Both primary and metastatic cholangiocarcinoma showed 100 percent binding to antibodies. Tissue specificity was Tumor cells and neoplastic biliary epithelium (Figure 11) There was no relationship between antibody binding and tumor stage, the antibody was strong against 9/9 non-neoplastic liver tissue sections The non-neoplastic liver tissue specificity was mainly to the bile duct epithelium and to a lesser extent also stained for hepatocytes.The AR59A816.14 antigen is a liver tumor tissue. Therefore, AR5 A816.14 have the potential as therapeutic agents in the treatment of liver cancer.

  Overwhelming evidence indicates that AR59A816.14 mediates anticancer effects through linkage with epitopes present in cancer cell lines and human tumor tissue. Furthermore, AR59A816.14 antibody can be used to detect cells and / or tissues that express epitopes that specifically bind using techniques exemplified by, but not limited to, FACS, cell ELISA or IHC. be able to.

  All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which this invention pertains. All patents and publications are incorporated herein by reference to the same extent as if each individual publication was clearly and individually indicated to be incorporated herein by reference.

  While specific forms of the invention have been illustrated, it should be understood that the invention is not limited to the specific forms or arrangements of parts described and shown herein. It will be apparent to those skilled in the art that various modifications can be made without departing from the scope of the invention and that the invention is not to be considered limited to what is shown and described in the specification. . Those skilled in the art will readily understand that the present invention is well adapted to carry out the objects and will readily obtain the objects and advantages described, as well as those inherent therein. Any of the oligonucleotides, peptides, polypeptides, biologically relevant compounds, methods, procedures and techniques described herein are presently representative of preferred implementations and are intended to be exemplary. And is not intended to limit the scope. Modifications and other uses of the specification will occur to those skilled in the art, which are encompassed within the spirit of the invention and are defined by the scope of the appended claims. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific examples. In fact, various modifications of the described modes for carrying out the invention will be apparent to those skilled in the art and are intended to be within the scope of the claims.

The patent or application file contains at least one drawing made in color. Copies of this patent or patent application publication in color drawings will be provided by the JPO upon request and payment of the necessary fees.
The cytotoxicity and binding level rates of hybridoma supernatants against cell lines MDA-MB-231, OVCAR-3, SW1116, Lovo and CCD-27sk are compared. FIG. 6 represents binding of AR59A816.14 and anti-EGFR control to cancer and normal cell lines. Data are summarized in a table representing the mean fluorescence intensity as an increase in folding over the isotype control. 1 includes representative FACS histograms of AR59A816.14 and anti-EGFR antibodies directed against several cancer and non-cancer cell lines. Figure 8 shows the effect of AR59A816.14 on tumor growth in a prophylactic Lovo colon cancer model. The vertical line indicates the period during which the antibody was administered. Data points represent the mean +/- SEM. Figure 3 shows the effect of AR59A816.14 on body weight in a prophylactic Lovo colon cancer model. Data points represent the mean +/- SEM. Figure 5 shows the effect of AR59A816.14 on tumor growth in a prophylactic DLD-1 colon cancer model. The vertical line indicates the period during which the antibody was administered. Data points represent the mean +/- SEM. Figure 3 shows the effect of AR59A816.14 on body weight in a prophylactic DLD-1 colon cancer model. Data points represent the mean +/- SEM. The table summarizes the IHC comparison of AR59A816.14 compared to positive and negative controls for human xenograft tumor tissue. AR59A816.14 for xenograft tumor tissue of breast MDA-MB-231 (A) or colon SW1116 (B), or buffer control for xenograft tumor tissue of breast MDA-MB-231 (C) or colon SW1116 (D) It is a typical micrograph which shows the coupling | bonding pattern. AR59A816.14 showed positive staining in tumor cells. The magnification is 400 ×. The table summarizes the IHC comparison of AR59A816.14 compared to positive and negative controls for human liver tumor microarrays. Representative photomicrographs showing the binding pattern of AR59A816.14 to primary (A) versus metastatic (B) HCC tumor tissue or buffer control to primary (C) versus metastatic (D) HCC tumor tissue It is. AR59A816.14 showed positive staining for metastasis at a higher rate compared to primary HCC samples. The magnification is 200 ×.

Claims (24)

  An isolated monoclonal antibody produced by a hybridoma deposited at IDAC with accession number 170505-02.   A humanized antibody produced from the isolated monoclonal antibody of claim 1.   A chimeric antibody produced from the isolated monoclonal antibody of claim 1.   An isolated hybridoma cell line deposited with IDAC under accession number 170505-02. A method for initiating antibody-induced cytotoxic activity of cancerous cells in a tissue sample selected from a human tumor comprising:
Providing a tissue sample from the human tumor;
An isolated monoclonal antibody produced by a hybridoma deposited at the IDAC with accession number 170505-02 or its CDMAB (where CDMAB has an ability to competitively inhibit the binding of said isolated monoclonal antibody to its target antigen). And contacting said tissue sample with said isolated monoclonal antibody or CDMAB thereof, and
Binding of the isolated monoclonal antibody or its CDMAB to the tissue sample induces cytotoxic activity
Method.   CDMAB of the isolated monoclonal antibody according to claim 1.   A CDMAB of the humanized antibody of claim 2.   CDMAB of the chimeric antibody according to claim 3.   9. The isolated antibody or CDMAB thereof according to any one of claims 1, 2, 3, 6, 7 or 8, which binds to a member selected from the group consisting of a cytotoxic moiety, an enzyme, a radioactive compound and a hematogenous cell. . A binding assay for determining the presence of cancerous cells in a tissue sample selected from a human tumor to which an isolated monoclonal antibody produced by hybridoma cell line AR59A816.14 having IDAC accession number 170505-02 specifically binds. ,
Providing a tissue sample from the human tumor;
Provided at least one isolated monoclonal antibody or CDMAB thereof that recognizes the same epitope or multiple epitopes recognized by the isolated monoclonal antibody produced by hybridoma cell line AR59A816.14 having IDAC accession number 170505-02 To do;
Contacting said tissue sample with said at least one isolated monoclonal antibody or its CDMAB; and determining binding of said at least one isolated monoclonal antibody or its CDMAB with said tissue sample;
Thereby indicating the presence of the cancerous cells in the tissue sample
Binding assay.   An isolated monoclonal antibody or its CDMAB encoded by a clone whose human tumor is deposited at IDAC with accession number 170505-02 (where CDMAB competitively binds the isolated monoclonal antibody to its target antigen) A method of treating the human tumor in a mammal that expresses at least one epitope or antigen that specifically binds to the mammal, characterized by the ability to inhibit, wherein the mammal is treated with the monoclonal antibody or CDMAB Administering in an amount effective to effect a reduction in tumor volume in said mammal.   12. The method of claim 11, wherein the isolated monoclonal antibody is conjugated to a cytotoxic moiety.   13. The method of claim 12, wherein the cytotoxic moiety is a radioisotope.   12. The method of claim 11, wherein the isolated monoclonal antibody or CDMAB thereof activates complement.   12. The method of claim 11, wherein the isolated monoclonal antibody or CDMAB thereof mediates antibody-dependent cytotoxic activity.   12. The method of claim 11, wherein the isolated monoclonal antibody is humanized.   12. The method of claim 11, wherein the isolated monoclonal antibody is chimerized.   An isolated monoclonal antibody or its CDMAB encoded by a clone whose human tumor is deposited at IDAC with accession number 170505-02 (where CDMAB competitively binds the isolated monoclonal antibody to its target antigen) A method of treating said human tumor that is sensitive to antibody-induced cytotoxic activity in a mammal that expresses at least one epitope or antigen that specifically binds (characterized by the ability to inhibit), said mammal And administering the monoclonal antibody or the CDMAB thereof in an amount effective to cause a reduction in tumor burden in the mammal.   19. The method of claim 18, wherein the isolated monoclonal antibody is conjugated to a cytotoxic moiety.   20. The method of claim 19, wherein the cytotoxic moiety is a radioisotope.   19. The method of claim 18, wherein the isolated monoclonal antibody or CDMAB thereof activates complement.   19. The method of claim 18, wherein the isolated monoclonal antibody or CDMAB thereof mediates antibody-dependent cytotoxic activity.   19. The method of claim 18, wherein the isolated monoclonal antibody is humanized.   19. The method of claim 18, wherein the isolated monoclonal antibody is chimerized.

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